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Related Experiment Video

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Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Published on: April 1, 2016

Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells.

Gaelen T Hess¹, Laure Frésard², Kyuho Han¹

¹Department of Genetics, Stanford University, Stanford, California, USA.

|November 8, 2016

Summary

This summary is machine-generated.

CRISPR-X enables precise protein engineering by repurposing cellular mutation machinery. This method generates targeted genetic variants in their natural context, advancing protein function studies and improvements.

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Area of Science:

Biotechnology
Molecular Biology
Protein Engineering

Background:

Directed evolution for protein engineering is often limited by global mutagenesis techniques.
Introducing DNA libraries for protein modification presents technical challenges.

Purpose of the Study:

To develop a novel strategy, CRISPR-X, for in situ protein engineering.
To leverage somatic hypermutation machinery for targeted mutagenesis.
To create diverse libraries of localized point mutations in endogenous genes.

Main Methods:

CRISPR-X utilizes catalytically inactive dCas9 fused to cytidine deaminase (AID) variants.
MS2-modified sgRNAs recruit AID variants to specific genomic loci.
Targeted mutagenesis of endogenous genes like GFP and PSMB5 was performed.
Hyperactive AID variants were used to mutagenize regions flanking transcriptional start sites.

Main Results:

CRISPR-X successfully generated localized point mutations with limited off-target effects.
Mutagenesis of GFP yielded spectrum-shifted variants, including EGFP.
Mutations conferring bortezomib resistance were identified in PSMB5.
Simultaneous targeting of multiple genomic locations and diverse loci was demonstrated.

Conclusions:

CRISPR-X offers a powerful approach for creating complex genetic variant libraries in native contexts.
This method is broadly applicable for investigating and enhancing protein function.
The strategy overcomes limitations of traditional directed evolution techniques.